Abstract Approximately half of the two million infections acquired at hospitals in the US are associated with implanted medical devices. Bacterial colonization of implanted medical device surfaces is a major cause of device failure and usually requires device removal coupled with long term antibiotic treatment. Risks of morbidity/mortality from surgery and prolonged hospitalization make early detection critical. However, detection is challenging at early stages prior to development of systemic symptoms of infection when bacteria localized to inaccessible regions of the implant. We will develop a novel medical imaging technique, X-ray excited luminescent chemical imaging (XELCI) to non-invasively detect and monitor bacterial biofilms on modified implant surfaces. Our long term goal is to develop a sensor to detect and monitor in vivo infection. The approach is innovative because no other imaging technique provides surface-specific chemical information at high-resolution through thick tissue. XELCI is a type of scanning optical microscopy wherein a narrow X-ray beam irradiates a radioluminescent film on the implant surface creating a local luminescent spot with pH -dependent spectra. Although the luminescence scatters and blurs as it passes through the tissue in the ?far field,? the spectrum depends upon the local pH at the luminescence source, and image resolution is defined by the X-ray beam width. We will apply the technique to detect acidosis beneath biofilms in order to detect and monitor infection. Our two specific aims are to 1) Refine and validate XELCI sensors in vitro and ex vivo; 2) Use XELCI to Assess pH Changes during Healing and Infection in a Rabbit Model. The proposed research is significant because it develops a non-invasive method to detect, monitor, and study biofilms in situ with the ultimate potential for reducing morbidity, mortality and associated cost from implant infections.